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United States Patent |
5,593,561
|
Cognard
,   et al.
|
January 14, 1997
|
Multiple electrophoresis method and apparatus for migration and transfer
of macromolecules
Abstract
A multiple electrophoresis method and apparatus for migrating and
transferring macromolecules in a vessel (10) containing a plurality of
parallel elongate electrodes (14), membranes (22) disposed vertically in
the vessel between the columns of electrodes (14), and an electrophoresis
gel which is inserted in the vessel in liquid form and which is
subsequently solidified to perform electrophoresis. When the
electrophoresis is completed, the gel is liquefied, dissolved, or
decomposed, and the membranes (22) onto which the macromolecules have been
transferred are withdrawn from the vessel.
Inventors:
|
Cognard; Dominique (Guyancourt, FR);
Hache; Jean (Voisin-le-Bretonneux, FR)
|
Assignee:
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Bertin & Cie (Plaisir, FR)
|
Appl. No.:
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464652 |
Filed:
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July 18, 1995 |
PCT Filed:
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December 2, 1993
|
PCT NO:
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PCT/FR93/01185
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371 Date:
|
July 18, 1995
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102(e) Date:
|
July 18, 1995
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PCT PUB.NO.:
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WO94/14526 |
PCT PUB. Date:
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July 7, 1994 |
Foreign Application Priority Data
Current U.S. Class: |
204/456; 204/462; 204/464 |
Intern'l Class: |
G01N 027/26 |
Field of Search: |
204/299 R,180.1,182.8,518
|
References Cited
U.S. Patent Documents
3888758 | Jun., 1975 | Saeed.
| |
4994166 | Feb., 1991 | Fernwood et al.
| |
Foreign Patent Documents |
313293 | Apr., 1989 | EP.
| |
826623 | Apr., 1938 | FR.
| |
WO90/02601 | Mar., 1990 | WO.
| |
WO90/05017 | May., 1990 | WO.
| |
Primary Examiner: Valentine; Donald R.
Assistant Examiner: Noguerola; Alexander
Attorney, Agent or Firm: Bell, Seltzer, Park & Gibson
Claims
We claim:
1. A multiple electrophoresis method for controlled migration of
macromolecules and transfer thereof onto membranes in a vessel containing
a plurality of parallel elongate electrodes and having means for applying
electrical potentials to the electrodes for establishing between them
successively a first electric field for macromolecule migration, and then
a second electric field perpendicular to the first for transferring the
macromolecules onto the membranes, the method comprising placing in the
vessel membranes which are parallel to one another and to the electrodes
and which extend between the electrodes, and filling the vessel with an
electrophoresis gel in the liquid phase, the filling being performed
before or after the membranes have been placed in the vessel, then
solidifying the gel, subsequently placing samples of macromolecules in
wells formed in the gel along one of the edges of the membranes, applying
electrical potentials to the electrodes initially to cause the
macromolecules to migrate through the gel, and then to transfer them onto
the membranes, then liquefying, dissolving, or decomposing the gel, and
removing the membranes from the vessel.
2. A method according to claim 1, further comprising emptying and cleaning
the vessel before a new electrophoresis operation.
3. A method according to claim 1 wherein the membranes to be placed in the
vessel are previously mounted on support and manipulation frames, and are
optionally tensioned on said frames.
4. A method according to claim 1 wherein the membranes to be placed in the
vessel form a continuous strip that is guided along a zigzag path over
parallel rollers.
5. A method according to claim 1 comprising heating or cooling the gel in
the vessel by means of the above-specified electrodes.
6. A method according to claim 1 wherein the electrodes are received in
microporous tubes and the method comprises causing a hot or cold liquid
electrolyte to circulate along said tubes to heat or cool the
electrophoresis gel.
7. A method according to claim 1 comprising surrounding the vessel in a
jacket for circulating a heat transfer liquid for heating and cooling the
electrophoresis gel.
8. A method according to claim 1 comprising dissolving the gel in the
vessel by injecting an appropriate enzymatical solution or in decomposing
it by applying suction, by injecting liquid under pressure, or by
mechanical or ultrasound vibration.
9. Multiple electrophoresis apparatus for controlled migration of
macromolecules and for transferring them onto membranes, the apparatus
comprising a vessel containing a plurality of parallel elongate electrodes
and having means for applying electrical potentials to the electrodes to
create between them successively a first electric field for macromolecule
migration and separation, and then a second electric field perpendicular
to the first for transferring the macromolecules onto the membranes, the
apparatus also comprising
means for guiding or supporting the membranes in the vessel parallel to one
another and to the above-specified electrodes;
means for filling the vessel with electrophoresis gel in the liquid phase;
means for solidifying the gel in the vessel; and
means for liquefying, dissolving, or decomposing the gel in the vessel.
10. Apparatus according to claim 9, including means for forming rows of
wells in the gel to receive samples of macromolecules.
11. Apparatus according to claim 9 wherein the means for liquefying the gel
in the vessel are of the type that raise temperature, by heating the
vessel and/or heating the electrodes and/or causing a hot liquid to
circulate along microporous tubes containing the electrodes, and/or in a
jacket surrounding the vessel.
12. Apparatus according to claim 9 wherein means for dissolving or
decomposing the gel are of a type comprising injecting a liquid under
pressure, or applying suction, or generating mechanical or ultrasound
vibrations.
13. Apparatus according to claim 9 wherein the membranes are fixed by their
edges on support frames optionally including means for tensioning the
membranes.
14. Apparatus according to claim 9 wherein the membranes form a continuous
strip that is guided along a zigzag path over rollers parallel to the
electrodes.
15. Apparatus according to claim 14, wherein the rollers for guiding said
strip are carried by a common support, together forming a moving unitary
assembly that can be inserted in the vessel and extracted therefrom.
16. Multiple electrophoresis apparatus for controlled migration of
macromolecules and for transferring them onto membranes, the apparatus
comprising a vessel containing a plurality of parallel elongate electrodes
and having means for applying electrical potentials to the electrodes to
create between them successively a first electric field for macromolecule
migration and separation, and then a second electric field perpendicular
to the first for transferring the macromolecules onto the membranes, the
apparatus also comprising
means for guiding or supporting the membranes in the vessel parallel to one
another and to the above-specified electrodes, wherein the membranes form
a continuous strip and the means for guiding or supporting the membranes
includes rollers parallel to the electrodes for guiding the strip along a
zigzag path, and wherein the rollers for guiding said strip are carried by
a common support, together forming a moving unitary assembly that can be
inserted in the vessel and extracted therefrom;
means for filling the vessel with electrophoresis gel in the liquid phase;
means for solidifying the gel in the vessel; and
means for liquefying, dissolving, or decomposing the gel in the vessel.
Description
The invention relates to a multiple electrophoresis method and apparatus
for migration and transfer of macromolecules, such as proteins or nucleic
acids.
It is now well known to separate samples of macromolecules by
electrophoresis, causing them to migrate through a plate of gel under the
effect of an electric field. The samples of macromolecules travel through
the gel over distances that are a function of their molecular masses.
Under the effect of a second electric field, perpendicular to the first
and to the plates of gel, it is possible to transfer the macromolecules
onto microporous membranes associated with the plates of gel.
That technique, which was initially lengthy and difficult to implement, has
been improved and simplified little by little so that it is now possible
to cause macromolecules to migrate and separate and then to transfer them,
all within the same vessel and using the same network of electrodes,
without any manipulation of the plates of gel or the membranes between
migration and transfer of the macromolecules.
In particular, international application WO 90/02601 in the name of the
Applicant describes a multiple electrophoresis vessel in which it is
possible to place a relatively large number of plates of gel associated
with membranes, in order to perform separation and transfer of
macromolecules in a manner that is automatic or quasi-automatic. In
addition, French patent application 91 08578 in the name of the Applicant
describes cassettes for making and manipulating plates of gel associated
with membranes, and suitable for use in the electrophoresis vessel
described in the above-mentioned international application.
Although those known means have made it possible to achieve results that
are quite remarkable, it remains relatively difficult to make up the units
each comprising a plate of gel plus a membrane, and special care is
required to ensure that the plates of gel are uniform and do not contain
bubbles of air, and to ensure that the membranes associated with said
plates of gel are thoroughly plane and adhere over their entire working
area to the plates of gel. Also, because the gel ages quickly and very
poorly, particularly when it is agarose, it is impossible to prepare large
quantities of plates of gel with membranes a long time in advance and to
store them for subsequent use.
A particular object of the present invention is to provide a solution to
the problems that is simple, effective, and cheap.
The invention provides a multiple electrophoresis method and apparatus for
separation of macromolecules in gel and transfer thereof onto membranes,
but without using prefabricated gel-membrane units.
The invention thus provides a multiple electrophoresis method for
controlled migration of macromolecules and transfer thereof onto membranes
in a vessel containing a plurality of parallel elongate electrodes and
having means for applying electrical potentials to the electrodes for
establishing between them successively a first electric field for
macromolecule migration, and then a second electric field perpendicular to
the first for transferring the macromolecules onto the membranes, the
method being characterized in that it consists in placing in the vessel
membranes which are parallel to one another and to the electrodes and
which extend between the electrodes, and in filling the vessel with an
electrophoresis gel in the liquid phase, the filling being performed
before or after the membranes have been placed in the vessel, then in
solidifying the gel, in subsequently placing samples of macromolecules in
wells formed in the gel along one of the edges of the membranes, in
applying electrical potentials to the electrodes initially to cause the
macromolecules to migrate through the gel, and then to transfer them onto
the membranes, then in liquefying, dissolving, or decomposing the gel, and
in removing the membranes from the vessel.
The invention thus makes it possible to avoid prefabricating plates of gel
before performing electrophoresis, thus achieving a considerable saving in
time, means, and effort.
The invention also improves the quality of the results obtained insofar as
electrophoresis takes place in a medium that is more uniform because there
are no longer any cassettes supporting plates of gel, thereby increasing
reliability and reproducibility of macromolecular separation and transfer.
In a first embodiment of the invention, the membranes for placing in the
vessel are mounted on frames for support and manipulation purposes, and
they are optionally lightly tensioned on the frames.
The frames serve solely to support and tension the membranes, and are much
simpler and more compact than in the prior art where the frames were used
for making and supporting plates of gel.
In addition, their use facilitates membrane manipulation.
In another embodiment of the invention, the membranes to be placed in the
vessel form a continuous strip that is guided along a zigzag path over
parallel rollers.
This further reduces and simplifies membrane manipulation both before and
after electrophoresis.
The gel used in the present invention is a gel (typically agarose) that can
be solidified and liquefied by varying temperature and/or concentration.
The invention also provides a multiple electrophoresis apparatus for
controlled migration of macromolecules and for transferring them onto
membranes, the apparatus comprising a vessel containing a plurality of
parallel elongate electrodes and having means for applying electrical
potentials to the electrodes to create between them successively a first
electric field for macromolecule migration and separation, and then a
second electric field perpendicular to the first for transferring the
macromolecules onto the membranes, the apparatus being characterized in
that it also comprises:
means for guiding or supporting the membranes in the vessel parallel to one
another and to the above-specified electrodes;
means for filling the vessel with electrophoresis gel in the liquid phase;
means for solidifying the gel in the vessel; and
means for liquefying, dissolving, or decomposing the gel in the vessel.
The means for liquefying the gel are of the type that raises temperature,
by heating the vessel and/or heating the electrodes, and/or by causing a
hot liquid to circulate in microporous tubes containing the electrodes.
The means for dissolving or decomposing the gel may, for example, be of the
type that injects liquid under pressure, or of the suction type, or else
of the ultrasound generator type.
In a first embodiment of this apparatus, the membranes are fixed by their
edges to support frames which optionally include means for tensioning the
membranes.
In a variant embodiment of the apparatus, the membranes form a continuous
strip that is guided along a zigzag path over rollers that are
substantially parallel to the electrodes.
Advantageously, the rollers for guiding the continuous strip are carried by
a common support and form a moving unitary assembly that can be inserted
into the vessel and removed therefrom.
The invention will be better understood and other characteristics, details,
and advantages thereof will appear more clearly on reading the following
description given by way of example and with reference to the accompanying
drawings, in which:
FIG. 1 is a diagrammatic vertical section view of electrophoresis apparatus
of the invention;
FIG. 2 is a fragmentary diagrammatic view in horizontal section on line
II--II of the FIG. 1 apparatus;
FIGS. 3, 4, and 5 are diagrammatic views of a membrane, a support frame,
and a well-forming comb;
FIG. 6 is a diagrammatic view in vertical section through a variant
embodiment of the apparatus of the invention;
FIG. 7 is a diagrammatic elevation view of a membrane support and guide
assembly, usable in the FIG. 6 apparatus; and
FIG. 8 is a fragmentary diagrammatic plan view of the FIG. 7 assembly.
Reference is made initially to FIGS. 1 to 5 which are diagrams of a first
embodiment of apparatus of the invention.
In FIG. 1, there can be seen an electrophoresis vessel 10 that is
substantially in the form of a rectangular parallelepiped, surrounded by a
jacket 12 through which a heat-conveying liquid circulates at a
temperature that can be regulated for the purpose of heating and cooling
the vessel 10.
As described in the Applicant's international application WO 90/02601, the
vessel preferably includes a plurality of elongate electrodes 14 which are
horizontal and parallel to one another, and which are organized in rows
and in columns to form a square-mesh array. The electrodes are constituted
by electrically-conductive metal wires and they are connected at their
ends (FIG. 2) to means 16 for applying electrical potentials so as to form
electric fields extending vertically or horizontally between the rows or
between the columns of electrodes.
In the embodiment shown in FIG. 1, the electrodes 14 are housed in small
tubes 18 of microporous material which form bubble traps and which may
also be used for circulating an appropriate electrolyte.
The vessel 10 also includes electrolyte filling and circulation means which
are represented diagrammatically in the form of two ducts 20 each
including opening and closing means such as a solenoid valve, one provided
on the bottom of the vessel 10 or in the vicinity of its bottom, and the
other at the top of the vessel.
According to the invention, these ducts 20 are used to fill the vessel with
an electrophoresis gel in liquid form, e.g. an agarose solution at a
temperature of the order of 50.degree. C. to 60.degree. C., approx. When
the vessel 10 is filled with the agarose solution, the gel is solidified,
either by allowing it to cool to ambient temperature, or by accelerating
the cooling by causing cold liquid to circulated in the jacket 12, or else
by causing cold electrolyte to circulate in the tubes 18 surrounding the
electrodes 14, or indeed by connecting the ends of the electrodes 14 to a
cold source (the electrodes 14 are generally made of platinum, which is a
very good conductor of heat).
In a variant, it is also possible to increase the concentration of the
agarose in the solution filling the vessel 10.
The vessel 10 then contains a large block of gel which can be used for
migration and separation of macromolecules, samples of which have been
deposited in wells formed in the top portion of the block of gel.
Naturally, it is necessary to place membranes of conventional type in the
vessel between the columns of electrodes, before proceeding with
solidification of the gel.
The simplest method of proceeding is doubtless to place vertical membranes
22 in the vessel between the rows of electrodes, the membranes 22 being,
for example, fixed to a support 24 via their top horizontal edges, and
being weighted down at their bottom horizontal edges. Thereafter the
vessel 10 is filled slowly from the bottom or the top, avoiding eddies, so
that the membranes remain as vertical as possible, after which the gel is
caused to solidify.
To make the membranes easier to manipulate, and also to guarantee that they
are plane when they are impregnated with gel in the liquid phase, it is
preferable to mount them on rigid support frames, proceeding essentially
in the manner described in the Applicant's international application WO
90/02601 and as shown diagrammatically in FIGS. 3 and 4. Under such
circumstances, two extensible strips 26 are initially stuck or welded to
the two longitudinal edges of a membrane 22, the strips including holes or
orifices 28 for fixing on pegs 30 that are guided to move along the
longitudinal edges of a frame 32 in the directions indicated by the
arrows. The pegs are urged resiliently outwards relative to the frame by
springs so as to keep the membrane 22 under light tension. The tension
serves to guarantee that the membrane 22 is plane and to take up any
stretching therein, due to impregnation by the liquid gel.
A strip 34 that includes a comb 36 along one of its edges can be engaged on
the frame 32 so that the comb 36 extends substantially along the top
horizontal edge of the membrane 22 so as to form, later on, wells in the
gel for receiving samples of macromolecules.
The apparatus of the invention is then used as follows:
Initially the membranes 22 are prepared by fixing the extensible strips 26
along their edges, and then placing the membranes 22 on the frames, and
finally installing the strips 34 that carry the combs 36.
Thereafter, the frames 32 together with the membranes 32 and the combs 36
are placed in the vessel as shown in FIG. 1, with the vessel being filled
with gel in the liquid phase either before or after the frames 32 and the
membranes 22 are installed, after which the gel is caused to solidify by
cooling and/or increasing its concentration of gelling agent.
Once the gel has solidified, the strips 34 carrying the combs 36 are
withdrawn and samples of macromolecules are deposited in the rows of wells
that are formed in the gel along the horizontal top edges of the membranes
22. Different electrical potentials are then applied to the rows of
electrodes 14 to create vertical electric fields between the rows of
electrodes, thereby causing the macromolecules contained in the
above-mentioned wells to move vertically through the gel towards the
bottom of the vessel 10.
After a certain amount of time, during which the macromolecules travel
distances through the gel that are a function of their molecular masses,
the distribution of electrical potentials at the electrodes 14 is altered
so as to apply different potentials to the columns of electrodes, thereby
establishing electric fields between them that are horizontal and
perpendicular to the membranes 22. Under the effect of these electric
fields, the macromolecules move horizontally through the gel and become
fixed on the membranes.
Thereafter, the gel is liquefied, dissolved, or decomposed.
This can be done by raising the temperature of the gel, by causing hot
water to flow in the jacket 12 surrounding the vessel 10. It can also be
done by causing hot electrolyte to flow along the tubes 18 containing the
electrodes 14. It is also possible to inject a hot liquid under pressure
into the vessel 10 to decompose the gel, or to dissolve it by injecting an
appropriate enzymatical liquid solution (e.g. an agarose solution for an
agarose gel).
In a variant, it is also possible to close the vessel 10 in leakproof
manner and establishes a partial vacuum therein, thereby causing water to
evaporate from the gel and thereby decompose the gel.
It is also possible to apply mechanical or ultrasound vibration to the gel,
thereby detaching the gel from the membranes and/or decomposing it more or
less completely.
Once the gel has been liquefied, dissolved, decomposed, or merely unstuck
from the membranes, the frames 32 are extracted from the vessel, the
membranes 22 are removed therefrom, and then processed in conventional
manner.
Thereafter, it is necessary to empty the vessel completely and to wash it
before filling it with a new gel in the liquid phase to perform a new
electrophoresis operation.
In the variant embodiment shown diagrammatically in FIGS. 6 to 8, the
membranes form a continuous strip 40 which is guided over top and bottom
horizontal rollers 42 and 44 mounted to rotate freely about horizontal
axes that are parallel to the electrodes 14 of the vessel. The top rollers
42 are above the electrodes 14 and the maximum level of gel in the vessel,
while the bottom rollers 44 are preferably below the bottom row of
electrodes 14. The strip 40 constituting the membranes passes
alternatively over a top roller 42, then a bottom roller 44, and then
another top roller 42, etc. so as to form vertical lengths of membrane
between successive columns of electrodes 14.
The rollers 42 and 44 are preferably carried by a common support, e.g.
comprising a top frame 46 that carries the axle of the rollers 42, and
vertical legs 48 attached to the top frame 46 and carrying the axles of
the bottom rollers 44. This common support is advantageously movable so as
to be capable of being extracted from the vessel 10 in order to position a
continuous strip of membrane 40 over the rollers 42 and 44, and then be
lowered into the vessel 10 for electrophoresis. The axles of at least some
of the top rollers 42 may be associated with return springs 50 as shown
diagrammatically in FIG. 7 so that the rollers 44 are urged continuously
upwards in order to exert a small amount of tension on the strip 40 that
is plunged in the vessel.
Otherwise, the apparatus can be identical to that described with reference
to FIGS. 1 to 5.
It is used as follows:
With the support of the rollers 42 and 44 withdrawn from the vessel, a
strip 40 is initially put into place over the rollers.
Simultaneously, the vessel 10 may be filled with gel in liquid form, after
which the roller support is lowered slowly into the vessel. Conversely, it
would also be possible to place the support inside the vessel before
filling it slowly with gel in liquid form.
Once the lengths of membrane have been completely immersed in the gel,
combs 34, 36 are placed between the top rollers 42 as shown
diagrammatically in FIG. 6. Like the rollers 42 and 44, the combs may be
carried by a common support.
Thereafter the gel is caused to solidify, using the means as described
above.
The combs are then removed and samples of macromolecules are deposited in
the wells formed in the gel by the teeth of the combs. Thereafter it
suffices to apply the desired potential differences to the electrodes 14
in order firstly to cause the macromolecules to migrate and to become
separated by moving vertically downwards, and then to cause the
macromolecules to be transferred horizontally onto the vertical lengths of
membrane.
The gel is then liquefied, dissolved, or decomposed using the same means as
those described above.
Thereafter the support for the rollers 42 and 44 can be removed from the
vessel and traction can be applied to one end of the strip so as to
recover the lengths of membrane onto which the macromolecules have been
transferred.
Simultaneously, new lengths of membrane for use in the following
electrophoresis operation are brought into place between the rollers 42
and 44.
During this time, the vessel 10 is emptied and cleaned and readied for a
new electrophoresis operation.
In a variant embodiment (not shown), the rollers for guiding the strip 40
may be vertical. In this case, it is preferable for the electrodes in the
vessel to be vertical likewise, so as to avoid excessively complicating
the placing of the lengths of strip between the planes of electrodes.
The continuous strip 40 which forms the lengths of membrane between the
planes of electrodes can be constituted by a long length of membrane
providing the membrane is mechanically strong enough. However, when the
mechanical characteristics of the membrane are weak, because of its
thinness or its material, it is preferable to fix predetermined lengths of
membrane over windows previously cut out in a support strip, which may be
made out of the same material as the membrane but of greater thickness, or
else out of a different material.
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